Chinese hamster ovary (CHO) cells are a preferred expression system for biopharmaceuticals like monoclonal antibodies (mAb). Sodium butyrate (NaBu) is known for increasing the cell specific productivity by altering product gene expression via epigenetic changes. Similar effects were recently found for retinoic acid (RA) . Therefore, unraveling specific underlying signaling events and variations on proteome level will provide a new basis for future engineering of production processes.
MAb producing CHO cells were cultivated in SILAC medium (light, medium, heavy lysine and arginine). 1 h and 24 h after agent addition (150 nM RA or 2 mM NaBu) cells were harvested for phosphoproteome as well as proteome analysis. A nuclear extraction was followed by a tryptic digest and SIMAC phosphopeptide enrichment. The obtained samples were analysed by nLC-ESI-orbitrap MS measurements. Finally, data evaluation was performed by Proteome Discoverer and in-house Fusion software.
Addition of NaBu resulted in increased productivity linked to decreased growth as observed with other CHO lines before [2,3]. Similar effects for RA were found accompanied by a 23 % higher product concentration. Statistical evaluation of MS data resulted in 256 significantly regulated phosphopeptides after NaBu and 407 by RA treatment. The data reveal a changed phosphorylation pattern of a variety of histone modifying enzymes like methyl- or acetyltransferases such as Suz12 and KAT8. These findings implicate also putative differences in transcriptional regulation of e.g. product genes. On proteome level almost 2000 nuclear proteins were quantified and 355 (NaBu) or 243 (RA) differentially expressed. KEGG-mapping revealed a broad coverage of RNA transport and spliceosome pathways.
The impact of both agents on CHO cells resulted in a temporary increased cell specific productivity. Retinoic acid induced changes in nuclear signaling and phosphorylation pattern of histone modifying enzymes similar to sodium butyrate next to effects on other interesting pathways like RNA transport.